1. Field of Invention
The present invention relates generally to a method of providing a unique cell culture medium. More particularly, this invention relates to a method of extracting fluid from a cell culture apparatus that includes a uniformly distributed extracellular matrix adhered to a substrate.
2. Background of Related Technology
The network of fibrous and globular proteins lying between cells is called the extra-cellular matrix (ECM). ECM is a vital component of the cellular environment. Various ECM components are secreted by cells to form an interstitial matrix and basement membrane, the framework to which cells are anchored in vivo. These structures provide spatial orientation and the stability required for the organization and development of tissue-specific histology. However, the ECM is not merely an inert scaffolding, but an essential player in the regulation of the cell growth and differentiation. The ECM provides a milieu which plays a pivotal role in regulating cellular functions during normal pathological remodeling processes such as embryonic development, tissue repair, inflammation, tumor invasion, and metastasis. For example, ECM is known to function in the induction, sequestration, storage and presentation of growth factors.
In recognition of the fact that ECM is a vital component of the cellular micro environment, more and more researchers are incorporating extracellular matrix into their cell culture systems. In vitro use of ECM provides cells with conditions which more closely approximate their in vivo physiologic environments. ECM provides cells with mechanical support and influences their behavior by providing biochemical cues that affect cells via cell surface receptors.
The basement membrane is a specific type of extracellular matrix and is composed primarily of laminin and type IV collagen. A well-known Basement Membrane Matrix extracted from the Engelbreth-Holm-Swarm mouse tumor, is sold under the brand name MATRIGEL®. The terms MATRIGEL®, MATRIGEL® Matrix, and the like, as used herein refer to BD MATRIGEL® Basement Membrane Matrix (Becton, Dickinson and Co.), a mixture of basement membrane proteins derived from the Engelbreth-Holm-Swarm mouse tumor. This mouse tumor is rich in basement membrane proteins. The major matrix components are laminin, collagen IV, entactin and heparan sulfate proteoglycan and also contains growth factors, matrix metalloproteinases (MMPs [collagenases]), and other proteinases (plasminogen activators), as well as several undefined compounds, but it does not contain any detectable levels of tissue inhibitors of metalloproteinases (TIMPs). At room temperature, MATRIGEL® Matrix gels to form reconstituted basement membrane and is similar in its structure, composition, physical property and ability to retain functional characteristics typical of basement membranes in vivo.
A number of methods have been developed using MATRIGEL® Matrix to investigate the invasion of the basement membrane matrix by tumor cells, in vitro. Typically these methods involve the coating of MATRIGEL® Matrix onto the microporous membranes of cell culture inserts. Conventional techniques to prepare an ECM containing cell culture system include, first, warming a cold, neutralized solution of soluble collagen to induce polymerization and precipitation of native fibrils. Incubating the MATRIGEL® Matrix at a raised temperature of over 4° C. polymerizes the matrix.
Whereas amorphous, chemically cross-linked and alkali denatured collagen films for use in cell cultures are often dried to improve shelf life and to eliminate the need to prepare the cell culture substrate prior to each use, cell culture substrates containing native fibrillar collagen are prepared and used only in the form of firm, adherent gels of native fibrils. These gels are most often produced, as mentioned above, by warming a cold, neutralized solution of soluble collagen to induce polymerization and precipitation of native fibrils. However, they are not dried for storage because previous attempts to collapse and dry the native fibrillar collagen gels have resulted in the loss of native structure, suboptimal fiber formation and poor permeability characteristics. Native fibrillar collagen cell culture substrates must, therefore, be made just prior to use. This increases the labor and inconvenience associated with studies involving cell cultures with native fibrillar collagen. Thus, there exists a need for a cell culture substrate that contains a native fibrillar collagen, such as those found in MATRIGEL® Matrix, that can be prepared from a manufacture well before their intended use.
Conventional methods of preparing a cell culture apparatus containing native fibrillar collagen desirably remove fluid residing in the matrix just prior to use and after the gel is polymerized. Conventional methods to remove the fluid from an ECM include air drying and drying at elevated temperatures. Common elevated drying conditions include rapid drying at elevated temperatures with a drying airflow which promote large salt crystals and more pronounced patterning of the MATRIGEL® Matrix. Rapid drying of the MATRIGEL® Matrix results in poor distribution of the invading cells.
Conventional fluid removing techniques also include placing the underside of the porous surface on an absorbent material for a period of 3 minutes to overnight and/or applying a gentle vacuum to the underside of the porous surface. Fluid removed by slow drying under conditions of slowly decreasing temperature and without airflow resulted in the most even coating and, thus, the most even distribution of invading cells. For example, Swiderek, et al (U.S. Pat. No. 5,731,417) discloses methods of making dried films of native fibrillar collagen for cell culture which includes air drying the substrates.
Conventional drying methods degrade the functionality of the cell culture apparatus. For example, conventional coating and drying methods often result in an uneven or disrupted coating which gives rise to uneven cell invasion manifested by the formation of the various patterns such as intense central dots or rings of invading cells. As a result, the accurate counting of invading cells under the microscope is greatly complicated. Additionally, the ability to discriminate between invasive and non-invasive cells is significantly diminished. Invasive cells include HT-1080 cells and non-invasive cells include NIH 3T3 cells. Therefore, a distributed fibrillar collagen substrate, such as a MATRIGEL® matrix, is desirably uniform and consistent.